Determination of Melatonin in Boiled Rice by HPLC

Abstract

A determination method of melatonin in boiled rice using precolumn derivatization was developed. The extraction method was as follows. Boiled rice (20 g), 10 g of distilled water and 0.1 g α-amylase were weighed into a mortar, the boiled rice was liquefied by homogenization at 40°C for 10 min, and stirred for 20 min on a magnetic stirrer. Then, 2.5 g of Na₂CO₃ was added to the mixture, the mixture was homogenized by a glass homogenizer, and stirred for 60 min at room temperature. The melatonin in the mixture was extracted with dichloromethane and adsorbed to Sep-Pak Silica cartridge. The adsorbed melatonin was eluted by dichloromethane/ methanol (1 : 1), and the eluant was evaporated to dryness. For the derivatization of melatonin, 150 μL of aqueous 2 mol/L Na₂CO₃, 100 μL of 100 mmol/L H₂O₂, 50 μL of 1 mmol/L CuSO₄ and 700 μL of water were added to the residue, and the mixture was heated in a water bath (94-96°C) for 6 min. The synthesized melatonin derivative was extracted with ethyl acetate, evaporated to dryness, dissolved in 15% (v/v) aqueous CH₃CN, and subjected to HPLC analysis. When standard melatonin was added to the liquefied boiled rice, the recovery rate was 99.6±6.9% (mean±S.D; n=6) for milled rice and 76.7±5.6% (n=4) for brown rice. Melatonin content in boiled rice was determined to be 71.6 and 86.2 pg/g for two cultivars of milled rice 107.0 and 130.2 pg/g for two cultivars of brown rice, and 36.2, 36.6 and 50.5 pg/g for three brands of commercial packaged boiled rice. These results suggest that the present method is suitable for the determination of melatonin in boiled rice.